title: Ubiquitination and degradation of CFTR by the E3 ubiquitin ligase MARCH2 through its association with adaptor proteins CAL and STX6.
abstract: Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.
authors.affiliation: Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Cheng
authors.forename: William
authors.initials: W
authors.lastname: Guggino
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D000077614:Golgi Matrix Proteins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D009154:Mutation; D011485:Protein Binding; D059748:Proteolysis / Q000187:drug effects; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C434484:GOPC protein, human; D000077614:Golgi Matrix Proteins; D018942:Macrolides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D050765:Qa-SNARE Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; C040929:bafilomycin A1; C502926:MARCHF2 protein, human; D044767:Ubiquitin-Protein Ligases